EP3515506B1 - Stilllegung von dux4 durch rekombinante geneditierungskomplexe - Google Patents

Stilllegung von dux4 durch rekombinante geneditierungskomplexe Download PDF

Info

Publication number
EP3515506B1
EP3515506B1 EP17853972.2A EP17853972A EP3515506B1 EP 3515506 B1 EP3515506 B1 EP 3515506B1 EP 17853972 A EP17853972 A EP 17853972A EP 3515506 B1 EP3515506 B1 EP 3515506B1
Authority
EP
European Patent Office
Prior art keywords
dux4
gene editing
subject
expression
recombinant gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP17853972.2A
Other languages
English (en)
French (fr)
Other versions
EP3515506A1 (de
EP3515506A4 (de
Inventor
Peter L. Jones
Charis L. HIMEDA
Takako JONES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Massachusetts UMass
Original Assignee
University of Massachusetts UMass
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Massachusetts UMass filed Critical University of Massachusetts UMass
Publication of EP3515506A1 publication Critical patent/EP3515506A1/de
Publication of EP3515506A4 publication Critical patent/EP3515506A4/de
Application granted granted Critical
Publication of EP3515506B1 publication Critical patent/EP3515506B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4707—Muscular dystrophy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • Facioscapulohumeral muscular dystrophy is caused by the aberrant expression of the DUX4 gene from an epigenetically dysregulated D4Z4 array at chromosome 4q35. This gene is generally not expressed, or expressed at very low levels, in healthy individuals. In FSHD patients, DUX4 is aberrantly expressed at higher levels in the skeletal muscles. This aberrant expression ultimately leads to muscle pathology, atrophy, and clinical weakness. Most therapies being developed target the DUX4 mRNA or protein.
  • Himeda et al., Trends Pharmacol. Sci., 37 (4), 249-251 compares the use of CRISPR/Cas9 genome editing tools in two myopathies with different molecular origins: Duchenne muscular dystrophy, a monogenetic disease, and FSHD, an epigenetic disorder with unique therapeutic challenges.
  • S151 discusses the role of DUX4 and FRG1 in FSHD, and the feasibility of RNAi therapy for dominant muscular dystrophies.
  • Wei et al., Mol. Ther., 17 (Suppl. 1), S200-201 (2009 ) discusses methods of targeting DUX4 by RNAi, and the effects on mouse muscle in a model of muscular dystrophy.
  • Wallace et al., Mol. Ther., 20 (7), 1417 - 1423 (2012 ) showed that adeno-associated viral (AAV) vector-delivered therapeutic microRNAs corrected DUX4- associated myopathy in mouse muscle.
  • AAV adeno-associated viral
  • compositions useful for the treatment of diseases associated with aberrant expression of DUX4 (e.g ., facioscapulohumeral muscular dystrophy, FSHD).
  • diseases associated with aberrant expression of DUX4 e.g ., facioscapulohumeral muscular dystrophy, FSHD.
  • compositions (e.g ., recombinant gene editing complexes) disclosed herein are useful because they transcriptionally regulate ( e.g ., inhibit) aberrant expression of DUX4.
  • reduction of DUX4 expression by compositions described by the disclosure in subjects having diseases characterized by aberrant expression of DUX4 (e.g., FSHD) is expected to result in decreased DUX4-fl expression (e.g. , reduced expression of the pathogenic DUX4-fl protein), and thereby decrease disease symptomatology or reverse disease symptoms.
  • DUX4-fl expression e.g., reduced expression of the pathogenic DUX4-fl protein
  • reduction of DUX4-fl expression by compositions (e.g ., gene editing complexes) described by the disclosure results in reduction of certain genes that function as downstream targets of DUX4, for example TRIM43 , ZSCAN4, and MBD3L2.
  • the disclosure provides a recombinant gene editing complex comprising: a recombinant gene editing protein; and, a nucleic acid encoding a guide RNA (gRNA) that specifically hybridizes to a target nucleic acid sequence encoding a D4Z4 macrosatellite repeat region, wherein binding of the complex to the target nucleic acid sequence results in inhibition of DUX4 gene expression.
  • gRNA guide RNA
  • the target nucleic acid sequence is located on chromosome 4 at position 4q35.
  • the gRNA comprises or is encoded by the sequence set forth in any one of SEQ ID NOs: 1-11. In some embodiments, the gRNA specifically hybridizes to a nucleic acid sequence encoding a DUX4 promoter or exon 1 of DUX4. In some embodiments, the gRNA comprises or is encoded by the sequence set forth in any one of SEQ ID NOs: 3-8. In some embodiments, the recombinant gene editing complex comprises a plurality ( e.g ., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of gRNA sequences (e.g ., a plurality of a single gRNA sequence, or a plurality of different or unique gRNA sequences).
  • a plurality e.g ., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
  • the recombinant gene editing protein comprises a Cas protein, a Cfp1 protein, or a variant thereof.
  • the Cas protein is a Streptococcus pyogenes Cas protein (SpCas) or a Staphylococcus aureus Cas protein (SaCas).
  • the Cas protein is a Cas9 protein or a dead Cas9 (dCas9) protein.
  • the recombinant gene editing protein comprises the sequence set forth in SEQ ID NO: 42 or 45.
  • the recombinant gene editing protein further comprises a transcriptional repressor domain.
  • the transcriptional repressor domain is a KRAB domain.
  • the gene editing protein comprises the sequence set forth in SEQ ID NO: 43 or 44.
  • the disclosure provides a vector comprising a nucleic acid encoding one or more components of a recombinant gene editing complex (e.g ., a nucleic acid encoding a gene editing protein, a gRNA, or both) as described by the disclosure.
  • the vector is a lentiviral vector or a recombinant adeno-associated virus vector (rAAV vector).
  • the disclosure provides a host cell comprising a nucleic acid encoding one or more components of a gene editing complex as described by the disclosure.
  • the disclosure provides a composition comprising a gene editing complex (e.g ., a nucleic acid encoding one or more components of a gene editing complex, or a vector comprising a nucleic acid encoding a gene editing complex) and a pharmaceutically acceptable excipient.
  • a gene editing complex e.g ., a nucleic acid encoding one or more components of a gene editing complex, or a vector comprising a nucleic acid encoding a gene editing complex
  • a pharmaceutically acceptable excipient e.g ., a nucleic acid encoding one or more components of a gene editing complex, or a vector comprising a nucleic acid encoding a gene editing complex
  • the disclosure relates, in part, to the discovery that, in some embodiments a gene editing complex as described by the disclosure is capable of reducing aberrant expression of DUX4.
  • the disclosure provides a method for inhibiting expression of DUX4 in a cell, the method comprising: contacting a cell with a nucleic acid encoding the recombinant gene editing complex as described by the disclosure, a vector as described by the disclosure, in an amount sufficient to inhibit expression of DUX4 gene in the cell.
  • the cell comprises a D4Z4 repeat array having 11 or less repeat units.
  • the cell is obtained from a subject having or at risk of having facioscapulohumeral muscular dystrophy (FSHD).
  • FSHD facioscapulohumeral muscular dystrophy
  • the cell is in vitro or ex vivo.
  • the disclosure provides a method for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of (i) a nucleic acid encoding the recombinant gene editing complex as described by the disclosure; or, (ii) a vector as described by the disclosure.
  • FSHD facioscapulohumeral muscular dystrophy
  • the subject is a mammal, optionally a human.
  • chromosome 4 of the subject comprises a D4Z4 repeat array having 11 or less repeat units.
  • the administration is by injection, optionally intramuscular injection or intravenous injection.
  • the recombinant gene editing complex is administered to muscle cells of the subject, optionally myogenic precursor satellite cells, myoblasts, myocytes, or terminally differentiated muscle cells of the subject.
  • the recombinant gene editing complex is administered to one or more somatic cells of the subject, optionally fibroblasts or leukocytes.
  • the one or more somatic cells are isolated from the subject and reprogrammed into a myogenic lineage, for example using induced pluripotent stem cell (iPS) technology.
  • iPS induced pluripotent stem cell
  • the disclosure relates to recombinant gene editing complexes useful for the treatment of diseases associated with aberrant expression of DUX4 (e.g ., facioscapulohumeral muscular dystrophy, FSHD).
  • DUX4 e.g ., facioscapulohumeral muscular dystrophy, FSHD.
  • the disclosure is based, in part, on the surprising discovery that recruitment of recombinant gene editing complexes to certain loci of the D4Z4 macrosatellite repeat region ( e.g ., at position 4q35) and/or the DUX4 gene results in decreased production of DUX4-fl, the pathogenic mRNA isoform associated with FSHD, as well as reduced expression of certain genes (e.g. , TRIM43 , ZSCAN4 , etc .) that are downstream targets of DUX4-fl.
  • certain genes e.g. , TRIM43 , ZSCAN4 , etc .
  • FSHD Facioscapulohumeral Muscular Dystrophy
  • facioscapulohumeral muscular dystrophy refers to a disease or disorder that results from transcriptional activation of the DUX4 gene.
  • activation of the DUX4 gene results in a toxic gain of function in the DUX4 gene (e.g ., production of pathogenic DUX4-fl) and causes a range of symptoms including progressive skeletal muscle weakness (e.g., facial muscle weakness, shoulder weakness, etc .), hearing loss, abnormal heart rhythm, unequal weakening of biceps, triceps, deltoids and lower arm muscles, loss of strength in abdominal and/or leg muscles, a foot drop.
  • FSHD results in a subject requiring ventilatory support or wheelchair confinement.
  • FSHD is associated with a contraction of the D4Z4 repeat sequence at the subtelomeric region 4q35 on Chromosome 4 of the human genome.
  • chromosome 4 of a healthy subject e.g ., a subject not having FSHD
  • the DUX4 gene of a subject having a contracted D4Z4 repeat becomes transcriptionally activated due to the loss of repeat-mediated repression, resulting in production of pathogenic full-length DUX4 (DUX4-fl) mRNA and protein.
  • the disclosure relates to the discovery that recruitment of recombinant gene editing molecules to certain loci of the D4Z4 macrosatellite repeat region and/or the DUX4 gene results in decreased production of DUX4-fl protein in a subject ( e.g ., in a cell of a subject).
  • D4Z4 macrosatellite repeat region refers to a chromosomal locus comprising at least one of the following genomic features: a p13E-11 probe hybridizing sequence, a non-deleted element (NDE) sequence (e.g ., a NDE sequence that functions as a transcriptional start site for a DBE-T transcript), a nucleic acid sequence encoding DUX4 (e.g ., a nucleic acid sequence encoding exon 1, exon 2 and exon 3 of DUX4).
  • NDE non-deleted element
  • DUX4 e.g a nucleic acid sequence encoding exon 1, exon 2 and exon 3 of DUX4
  • chromosome 4 comprises a D4Z4 macrosatellite repeat region, for example at locus 4q35.
  • the term "reduced DUX4 expression” refers to a change in state of a DUX4 gene from a transcriptionally active (e.g ., expressed or transcribed) state to a reduced state of transcriptional activity, for example, a transcriptionally inactive (e.g ., silenced) state.
  • a subject e.g. , a cell in a subject
  • a transcriptionally active (e.g., expressed) DUX4 gene produces DUX4-fl; knockdown (e.g., silencing) of DUX4 expression in the subject ( e.g. , cell in the subject), for example by administration of a recombinant gene editing complex targeting a D4Z4 macrosatellite repeat region, leads to reduced (or inhibited) expression and production of DUX4-fl in the subject.
  • the disclosure relates to the discovery that inhibition of DUX4 expression by a recombinant gene editing complex as described by the disclosure results in reduced expression of certain genes that are downstream targets of DUX4-fl.
  • inhibition of DUX4 expression by a recombinant gene editing complex as described by the disclosure results in reduced expression of TRIM43, ZSCAN4, MBD3L2, or any combination of the foregoing, as described further in the Examples section.
  • reduction of target gene (e.g ., DUX4 , TRIM43 , ZSCAN4 ) expression can be measured as expression level of the target gene (e.g ., DUX4) in a sample ( e.g ., a cell or a subject) after treatment with a recombinant gene editing complex relative to expression level of target gene ( e.g ., DUX4) in the sample prior to treatment with the recombinant gene editing complex.
  • the expression level of a target gene can be measured by any suitable method known in the art, for example by hybridizationbased assay (e.g ., RT-PCR, qRT-PCR, Northern Blot), protein-based methods (e.g ., Western blot), spectroscopic methods (e.g ., mass spectrometry), and cell-based methods (e.g ., flow cytometry, fluorescence activated cell sorting (FACS)).
  • hybridizationbased assay e.g ., RT-PCR, qRT-PCR, Northern Blot
  • protein-based methods e.g ., Western blot
  • spectroscopic methods e.g ., mass spectrometry
  • cell-based methods e.g ., flow cytometry, fluorescence activated cell sorting (FACS)
  • the disclosure is based, in part, on the discovery of recombinant gene editing complexes that inhibit-for example, through transcriptional repression or generation of double-stranded DNA breaks- expression of the DUX4 gene.
  • the disclosure provides a recombinant gene editing complex comprising: a recombinant gene editing protein; and, a nucleic acid encoding a guide RNA (gRNA) that specifically hybridizes to a target nucleic acid sequence encoding a D4Z4 macrosatellite repeat region, wherein binding of the complex to the target nucleic acid sequence results in inhibition of DUX4 gene expression.
  • gRNA guide RNA
  • gene editing complex refers to a biologically active molecule (e.g ., a protein, one or more proteins, a nucleic acid, one or more nucleic acids, or any combination of the foregoing) configured for adding, disrupting or changing genomic sequences (e.g ., a gene sequence), for example by causing a double stranded break (DSB) in a target DNA or inhibiting transcription of a target DNA sequence.
  • a biologically active molecule e.g ., a protein, one or more proteins, a nucleic acid, one or more nucleic acids, or any combination of the foregoing
  • genomic sequences e.g ., a gene sequence
  • gene editing complexes include but are not limited to Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), engineered meganuclease re-engineered homing endonucleases, the CRISPR/Cas system, and meganucleases (e.g. , Meganuclease I-SceI).
  • TALENs Transcription Activator-like Effector Nucleases
  • ZFNs Zinc Finger Nucleases
  • meganucleases e.g. , Meganuclease I-SceI
  • a gene editing complex comprises proteins or molecules (e.g ., recombinant gene editing proteins) related to the CRISPR/Cas system, including but not limited to Cas9,Cas6, dCas9, Cpf1, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and variants thereof.
  • proteins or molecules e.g ., recombinant gene editing proteins related to the CRISPR/Cas system, including but not limited to Cas9,Cas6, dCas9, Cpf1, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and variants thereof.
  • a recombinant gene editing protein is a nuclease.
  • nuclease refers to an enzyme that cleaves a phosphodiester bond or bonds within a polynucleotide chain.
  • Nucleases may be naturally occurring or genetically engineered. Genetically engineered nucleases are particularly useful for genome editing and are generally classified into four families: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (e.g ., engineered meganucleases) and CRISPR-associated proteins (Cas nucleases).
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • meganucleases e.g ., engineered meganucleases
  • CRISPR-associated proteins Cas nucleases
  • the nuclease is a ZFN. In some embodiments, the ZFN comprises a FokI cleavage domain. In some embodiments, the ZFN comprises Cys 2 His 2 fold group. In some embodiments, the nuclease is a TALEN. In some embodiments, the TALEN comprises a FokI cleavage domain. In some embodiments, the nuclease is a meganuclease. Examples of meganucleases include but are not limited to I-SceI, I-CreI, I-Dmol, and combinations thereof ( e.g ., E-DreI, DmoCre).
  • CRISPR refers to "clustered regularly interspaced short palindromic repeats", which are DNA loci containing short repetitions of base sequences. CRISPR loci form a portion of a prokaryotic adaptive immune system that confers resistance to foreign genetic material. Each CRISPR loci is flanked by short segments of "spacer DNA”, which are derived from viral genomic material. In the Type II CRISPR system, spacer DNA hybridizes to transactivating RNA (tracrRNA) and is processed into CRISPR-RNA (crRNA) and subsequently associates with CRISPR-associated nucleases (Cas nucleases) to form complexes that recognize and degrade foreign DNA.
  • tracrRNA transactivating RNA
  • crRNA CRISPR-RNA
  • Cas nucleases CRISPR-associated nucleases
  • the nuclease is a CRISPR-associated nuclease (Cas nuclease).
  • CRISPR nucleases include, but are not limited to Cas9, dCas9, Cas6, Cpf1, and variants thereof.
  • the nuclease is Cas9.
  • the Cas9 is derived from the bacteria Streptococcus pyogenes ( e.g., SpCas9) or Staphylococcus aureus ( e.g., SaCas9, for example SEQ ID NO: 42).
  • a Cas protein is modified ( e.g . genetically engineered) to lack nuclease activity.
  • dead Cas9 (dCas9) protein binds to a target locus but does not cleave said locus.
  • a dCas9 protein comprises the sequence set forth in SEQ ID NO: 45.
  • a Cas protein or variant thereof does not exceed the packaging capacity of a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector, for example as described by Ran et al. (2015) Nature. 520(7546); 186-91 .
  • a nucleic acid encoding a Cas protein is less than about 4.6 kb in length.
  • a catalytically dead Cas9 protein e.g ., dead Cas9, "dCas9"
  • a transcriptional regulator domain e.g ., covalently bound
  • a target gene e.g., DUX4
  • dCas9 comprises a sequence set forth in SEQ ID NO: 45.
  • dCas9 or another catalytically dead Cas protein
  • mediates transcriptional repression in some embodiments, by sterically hindering the binding of transcriptional machinery (e.g ., a RNA polymerase complex) to a target sequence.
  • transcriptional machinery e.g ., a RNA polymerase complex
  • a CRISPR Cas protein e.g ., dCas9 is fused to a transcriptional regulator domain.
  • a "transcriptional regulator domain” is a protein domain that catalyzes structural or chemical changes in a chromatin molecule that results in altered transcriptional activity (e.g ., transcriptional activation or transcriptional repression).
  • the transcriptional regulator domain is a transcriptional repressor domain.
  • the repressive domain comprises a Kruppel associated box domain (KRAB domain).
  • KRAB domains include KOX1 KRAB domain, KOX8 KRAB domain, ZNF43 KRAB domain, and ZNF184 KRAB domain.
  • the KRAB domain is a KOX1 KRAB domain.
  • the gene editing protein comprises a sequence set forth in SEQ ID NO: 43 or 44.
  • repressive domains include Chromo Shadow (CS) domain (e.g ., CS domain of HP1 ⁇ ) and WRPW domain (e.g ., WRPW domain of Hes1).
  • the transcriptional regulator domain is a transcriptional activator domain.
  • the transcriptional activator domain comprises a transcriptional activation domain of Herpes simplex virus, such as VP16, or a variant thereof.
  • a transcription factor may contain multiple activation domains, for example as described by Beerli et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95(25); 14628-33 .
  • the transcriptional activator domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 activation domains.
  • the transcriptional activator domain is VP64, VP96, or VP160.
  • a Cas protein is not fused to a transcriptional regulator domain, and is capable of modulating (e.g ., inhibiting) gene expression via nuclease activity (e.g ., DNA cleavage).
  • Cas9 cleaves DNA at a site targeted by the guide RNA and then repaired by either non-homologous end joining (NHEJ), which is imprecise and often results in a small insertion or deletion (InDel) that disrupts the targeted sequence, or homology directed DNA repair, which allows for the insertion of a changed or new DNA sequence into the genome at a specific location.
  • NHEJ non-homologous end joining
  • InDel small insertion or deletion
  • homology directed DNA repair which allows for the insertion of a changed or new DNA sequence into the genome at a specific location.
  • the disclosure relates to a gene editing complex comprising a functional nuclease and a guide RNA that hybridizes to a D4Z4 macrosatellite repeat region that is capable of reducing expression of DUX4 in a subject ( e.g ., a cell of a subject).
  • the guide RNA that directs the activity of the functional nuclease targets exon 3 of the DUX4 gene.
  • the CRISPR system can be modified to combine the tracrRNA and crRNA in to a single guide RNA (sgRNA) or just (gRNA).
  • sgRNA single guide RNA
  • gRNA guide RNA
  • gRNA guide RNA
  • sgRNA single guide RNA
  • gRNA gRNA
  • sgRNA a polynucleotide sequence that is complementary to a target sequence in a cell and associates with a Cas nuclease, thereby directing the Cas nuclease to the target sequence.
  • a gRNA e.g., sgRNA
  • a gRNA ranges between 5 and 25 nucleotides in length.
  • a gRNA ranges between 10 and 22 nucleotides in length. In some embodiments, a gRNA (e.g., sgRNA) ranges between 14 and 24 nucleotides in length.
  • a Cas protein and a guide RNA e.g ., sgRNA
  • a Cas protein and a guide RNA are expressed from the same vector. In some embodiments, a Cas protein and a guide RNA (e.g ., sgRNA) are expressed from separate vectors ( e.g ., two or more vectors).
  • a guide RNA e.g., a gRNA or sgRNA hybridizes (e.g ., binds specifically to, for example by Watson-Crick base pairing) to a target sequence and thus directs the CRISPR/Cas protein to the target sequence.
  • a guide RNA hybridizes to (e.g., targets) a nucleic acid sequence encoding a D4Z4 macrosatellite repeat region or a nucleic acid sequence encoding DUX4 gene.
  • the gRNA hybridizes to a p13E-11 sequence or a NDE sequence.
  • the gRNA hybridizes to exon 1, exon 2 or exon 3 of a DUX4 gene.
  • the gRNA comprises or is encoded by the sequence set forth in any one of SEQ ID NOs: 1-11, for example as described in Table 1.
  • the disclosure provides methods for treating a subject having facioscapulohumeral muscular dystrophy (FSHD).
  • FSHD facioscapulohumeral muscular dystrophy
  • transcriptional activation of the DUX4 gene may lead to FSHD in a subject.
  • a "subject" is interchangeable with a "subject in need thereof", both of which may refer to a subject having FSHD, or a subject having an increased risk of developing such a disorder relative to the population at large.
  • a subject has a D4Z4 array comprising 11 or fewer repeat units at chromosome 4q35 but does not exhibit signs or symptoms of FSHD.
  • a subject in need thereof may be a subject having a transcriptionally active DUX4 gene (e.g ., a subject expressing DUX4-fl protein).
  • a subject can be a human, non-human primate, rat, mouse, cat, dog, or other mammal.
  • treatment refers to therapeutic treatment and prophylactic or preventative manipulations.
  • the terms further include ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying causes of symptoms, preventing or reversing causes of symptoms, for example, symptoms associated with FSHD.
  • the terms denote that a beneficial result has been conferred on a subject having FSHD, or with the potential to develop such a disorder.
  • treatment is defined as the application or administration of an agent (e.g ., therapeutic agent or a therapeutic composition) to a subject, or an isolated tissue or cell line from a subject, who may have a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
  • an agent e.g ., therapeutic agent or a therapeutic composition
  • Therapeutic agents or therapeutic compositions may include a compound in a pharmaceutically acceptable form that prevents and/or reduces the symptoms of a particular disease (e.g ., FSHD).
  • a therapeutic composition may be a pharmaceutical composition that prevents and/or reduces the symptoms of FSHD.
  • the disclosure provides a composition (e.g ., a therapeutic composition) comprising a gene editing complex as described by the disclosure or a vector as described by the disclosure.
  • the composition further comprises a pharmaceutically acceptable excipient. It is contemplated that the therapeutic composition of the present invention will be provided in any suitable form. The form of the therapeutic composition will depend on a number of factors, including the mode of administration as described herein.
  • the therapeutic composition may contain diluents, adjuvants and excipients, among other ingredients as described herein.
  • the disclosure provides a method for inhibiting (e.g ., silencing) a transcriptionally active DUX4 gene in a cell, the method comprising: delivering to the cell an effective amount of gene editing complex, wherein the gene editing complex reduces DUX4 expression in the cell.
  • the cell containing an effective amount of a gene editing complex can be any cell that has a transcriptionally active DUX4 gene.
  • the cell can be a muscle cell (e.g ., a myoblast, myocyte, or terminally differentiated muscle cell).
  • a cell having a transcriptionally active DUX4 gene can also comprise a contraction of a D4Z4 repeat array at chromosome 4q35 of the DUX4 gene.
  • the number of repeat units in the array can vary. In some embodiments, the number of repeat units in the contraction is 11 or fewer ( e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0) repeat units. In some embodiments, the cell is in vitro or ex vivo.
  • ex vivo modified cell refers to a cell (e.g ., a mammalian cell) that is removed from a subject, genetically modified (e.g ., transfected or transduced with exogenous nucleic acids, or genetically reprogrammed), cultured or expanded, and optionally, returned to a subject ( e.g ., either the same subject, or a different subject).
  • ex vivo modified cells are useful for autologous cell therapy, or allogeneic cell therapy.
  • cells may be removed from a subject having a disease associated with a particular genetic defect (e.g ., FSHD), transfected with a gene editing complex that corrects the genetic defect (e.g . reduces expression of DUX4), and reintroduced into the subject.
  • a gene editing complex that corrects the genetic defect (e.g . reduces expression of DUX4)
  • cells are removed from a subject, genetically reprogrammed (e.g ., dedifferentiated or transdifferentiated into muscle cells), expanded, and reintroduced into the subject.
  • ex vivo modified cells produced by transfection with a nucleic acid as described by the disclosure have an improved safety profile compared to ex vivo cells produced by currently available gene therapy vectors.
  • the disclosure relates to pharmaceutical compositions comprising a gene editing complex.
  • the composition comprises gene editing complex and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Pharmaceutical compositions can be prepared as described below.
  • the active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient.
  • the compositions may be sterile.
  • compositions are formulated for delivering an effective amount of an agent (e.g ., gene editing complex).
  • an "effective amount" of an active agent refers to an amount sufficient to elicit the desired biological response (e.g., transcriptional repression, such as silencing or inhibition, of the active DUX4 gene).
  • An effective amount of an agent may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated (e.g ., FSHD), the mode of administration, and the patient.
  • a composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known in the art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990 ).
  • any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agent may be utilized for preparing and administering the pharmaceutical compositions of the present disclosure.
  • Illustrative of such methods, vehicles and carriers are those described, for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970 ).
  • Those skilled in the art, having been exposed to the principles of the disclosure, will experience no difficulty in determining suitable and appropriate vehicles, excipients and carriers or in compounding the active ingredients therewith to form the pharmaceutical compositions of the disclosure.
  • an effective amount, also referred to as a therapeutically effective amount, of a compound is an amount sufficient to ameliorate at least one adverse effect associated with activation (e.g ., transcriptional activation), or increased expression, of the gene in a cell or in an individual in need of such modulation.
  • an effective amount is an amount sufficient to inhibit ( e.g ., transcriptionally repress) DUX4 gene in a cell or in an individual in need of DUX4 inhibition.
  • the therapeutically effective amount to be included in pharmaceutical compositions depends, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, etc.
  • an amount of active agent is included in each dosage form to provide from about 0.1 to about 250 mg/kg, and preferably from about 0.1 to about 100 mg/kg.
  • an amount of active agent can be included in each dosage form to provide between about 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per subject.
  • One of ordinary skill in the art would be able to determine empirically an appropriate therapeutically effective amount.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular therapeutic agent being administered, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular nucleic acid and/or other therapeutic agent without necessitating undue experimentation.
  • colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a colloidal system of the disclosure is a liposome.
  • Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vesicles (LUVs), which range in size from 0.2 - 4.0 ⁇ m can encapsulate large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form. Fraley et al. (1981) Trends Biochem Sci 6:77 .
  • Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • Ligands which may be useful for targeting a liposome to, for example, an smooth muscle cell include, but are not limited to: intact or fragments of molecules which interact with smooth muscle cell specific receptors and molecules, such as antibodies, which interact with the cell surface markers of cancer cells. Such ligands may easily be identified by binding assays well known to those of skill in the art.
  • the liposome may be targeted to a tissue by coupling it to an antibody known in the art.
  • Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENE TM (a non-liposomal lipid with a special DNA condensing enhancer) and SUPERFECT TM (a novel acting dendrimeric technology).
  • EFFECTENE TM a non-liposomal lipid with a special DNA condensing enhancer
  • SUPERFECT TM a novel acting dendrimeric technology
  • Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN TM and LIPOFECTACE TM , which are formed of cationic lipids such as N-[1-(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • LIPOFECTIN TM and LIPOFECTACE TM are formed of cationic lipids such as N-[1-(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA N-[1-(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride
  • DDAB dimethyl dioctadecylammonium bromide
  • Certain cationic lipids including in particular N-[1-(2, 3 dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), may be advantageous when combined with the gene editing complexes and vectors of the disclosure.
  • DOTAP N-[1-(2, 3 dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate
  • compaction agents may also be desirable.
  • Compaction agents also can be used alone, or in combination with, a biological or chemical/physical vector.
  • a "compaction agent”, as used herein, refers to an agent, such as a histone, that neutralizes the negative charges on the nucleic acid and thereby permits compaction of the nucleic acid into a fine granule. Compaction of the nucleic acid facilitates the uptake of the nucleic acid by the target cell.
  • the compaction agents can be used alone, e.g., to deliver a gene editing complex or a vector as described by the disclosure in a form that is more efficiently taken up by the cell or, in combination with one or more of the above-described carriers.
  • the compounds may be administered alone (e.g ., in saline or buffer) or using any delivery vehicle known in the art.
  • delivery vehicles have been described: cochleates; Emulsomes; ISCOMs; liposomes; live bacterial vectors (e.g ., Salmonella, Escherichia coli, Bacillus Calmette-Guérin, Shigella, Lactobacillus); live viral vectors (e.g ., Vaccinia, adenovirus, Herpes Simplex, Lentiviral); microspheres; nucleic acid vaccines; polymers ( e.g ., carboxymethylcellulose, chitosan); polymer rings; proteosomes; sodium fluoride; transgenic plants; virosomes; and, virus-like particles.
  • gene editing complexes described by the disclosure are delivered by lentiviral vector or recombinant adeno-associated virus (rAAV) vector.
  • compositions of the disclosure are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • the compounds may also be formulated as a depot preparation.
  • Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin.
  • the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R (1990) Science 249:1527-1533 .
  • the compounds may be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
  • Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic.
  • such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories.
  • compositions of the present disclosure preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally as a tablet, capsule or pill, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, or transdermally.
  • a therapeutically effective amount of a gene editing complex or vector as described by the disclosure is delivered to a target tissue or a target cell.
  • DUX4 e.g. , DUX4-fl
  • an effective amount of gene editing complex or vector is delivered to the muscle cells of a subject.
  • the muscle cells are myoblasts.
  • the muscle cells are terminally differentiated muscle cells. Examples of differentiated muscle cells include myocytes and myotubes.
  • compositions containing gene editing complex or vector, and/or other compounds can be administered by any suitable route for administering medications.
  • a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular agent or agents selected, the particular condition being treated, and the dosage required for therapeutic efficacy.
  • the methods of this disclosure may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic effect without causing clinically unacceptable adverse effects.
  • modes of administration are discussed herein.
  • an effective amount of the gene editing complex or vector, and/or other therapeutic agent can be administered to a subject by any mode that delivers the agent to the desired surface, e.g ., systemic, intramuscular, etc.
  • the gene editing complex or vector as described by the disclosure is administered to a subject via intramuscular (IM) injection.
  • IM intramuscular
  • administering the pharmaceutical composition of the present disclosure may be accomplished by any means known to the skilled artisan.
  • Additional routes of administration include but are not limited to oral, parenteral, intravenous, intraperitoneal, intranasal, sublingual, intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal.
  • Systemic routes include oral and parenteral.
  • Several types of devices are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.
  • MDI metered dose inhalers
  • DPI dry powder inhaler
  • spacer/holding chambers in combination with MDI and nebulizers.
  • the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
  • the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g ., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active compounds may be in powder form for constitution with a suitable vehicle, e.g ., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g ., sterile pyrogen-free water
  • the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g ., containing conventional suppository bases such as cocoa butter or other glycerides.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109 .
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides
  • hydrogel release systems silastic systems
  • peptide-based systems such as wax, di-, and tri-glycerides
  • wax coatings such as those described in U.S. Pat. Nos.
  • a gene editing complex (e.g ., a nucleic acid encoding one or more components of a gene editing complex) can be delivered to the cells via an expression vector engineered to express the gene editing complex.
  • An expression vector is one into which a desired sequence may be inserted, e.g ., by restriction and ligation, such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
  • An expression vector typically contains an insert that is a coding sequence for a protein (e.g., gene editing protein, such as a CRISPR/Cas protein) or for a polynucleotide, such as guide RNA (gRNA, sgRNA, etc .).
  • Vectors may further contain one or more marker sequences suitable for use in the identification of cells that have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detectable by standard assays or fluorescent proteins, etc.
  • a coding sequence e.g ., protein coding sequence, miRNA sequence, shRNA sequence
  • regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • coding sequences be translated into a functional protein
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • a coding sequence may encode an miRNA, shRNA or siRNA.
  • the precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • Such 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene.
  • a vector does not include a promoter sequence.
  • Regulatory sequences may also include enhancer sequences, upstream activator sequences, internal ribosomal entry sites (IRES), and/or self-processing peptide sequences (e.g ., 2A peptide), as desired.
  • the vectors of the disclosure may optionally include 5' leader or signal sequences.
  • a virus vector for delivering a nucleic acid molecule is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccinia viruses and attenuated poxviruses, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty virus-like particle.
  • viruses and virus-like particles which have been used to deliver exogenous nucleic acids include: replication-defective adenoviruses, a modified retrovirus, a nonreplicating retrovirus, a replication defective Semliki Forest virus, canarypox virus and highly attenuated vaccinia virus derivative, non-replicative vaccinia virus, replicative vaccinia virus, Venzuelan equine encephalitis virus, Sindbis virus, lentiviral vectors and Ty virus-like particle.
  • the adeno-associated virus is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • a recombinant AAV vector comprises, at a minimum, a transgene coding sequence (e.g., a nucleic acid sequence encoding a gene editing protein, such as a Cas protein, or a gRNA) and its associated regulatory sequence flanked by two AAV inverted terminal repeat (ITR) sequences.
  • a transgene coding sequence e.g., a nucleic acid sequence encoding a gene editing protein, such as a Cas protein, or a gRNA
  • ITR inverted terminal repeat
  • the ITR sequences are AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9 ITR sequences, or variants thereof.
  • an rAAV vector comprising a nucleic acid encoding all or part of a gene editing complex (e.g., a nucleic acid sequence encoding a gene editing protein, a gRNA, or both) is packaged into a recombinant AAV (rAAV).
  • a gene editing complex e.g., a nucleic acid sequence encoding a gene editing protein, a gRNA, or both
  • rAAV recombinant AAV
  • an AAV vector is packaged into viral particles by one or more AAV capsid proteins.
  • the AAV capsid is an important element in determining these tissue-specific targeting capabilities.
  • an rAAV having a capsid appropriate for the tissue being targeted can be selected.
  • the capsid protein has a serotype selected from AAV2, AAV3, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.10, AAVrh.39, and AAVrh.43.
  • the rAAV comprises a capsid protein that targets muscle cells.
  • Non-cytopathic viruses include certain retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • the retroviruses are replication-deficient (e.g., capable of directing synthesis of the desired transcripts, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • gene editing complex e.g ., a nucleic acid sequence encoding a gene editing protein, a gRNA, or both
  • a cell e.g . a cell of a subject
  • nucleic acid molecules of the disclosure may be introduced into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host.
  • Such techniques include transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid molecule of interest, liposome-mediated transfection, and the like.
  • N-TER TM Nanoparticle Transfection System by Sigma-Aldrich FectoFly TM transfection reagents for insect cells by Polyplus Transfection, Polyethylenimine "Max” by Polysciences, Inc., Unique, Non-Viral Transfection Tool by Cosmo Bio Co., Ltd., Lipofectamine TM LTX Transfection Reagent by Invitrogen, SatisFection TM Transfection Reagent by Stratagene, Lipofectamine TM Transfection Reagent by Invitrogen, FuGENE ® HD Transfection Reagent by Roche Applied Science, GMP compliant in vivo- jetPEI TM transfection reagent by Polyplus Transfection, and Insect GeneJuice ® Transfection Reagent by Novagen.
  • pHAGE EFl-dCas9-VP64 (Addgene plasmid #50918), pHAGE EF1-dCas9-KRAB (Addgene plasmid#50919), pLKO.1-puro U6 sgRNA BfuAI stuffer (Addgene plasmid #50920), pHRdSV40-dCas9-10xGCN4-v4-P2A-BFP (Addgene plasmid #60903), and pHRdSV40-scFv-GCN4-sfGFP-VP64-GB 1-NLS (Addgene plasmid #60904), pHR-scFv-GCN4-sfGFP-GB 1-NLS-dWPRE (Addgene plasmid #60906).
  • ChIP-grade antibodies used in this example include: ⁇ -KAP1(ab3831), ⁇ -HP1 ⁇ (ab77256), ⁇ -HP1 ⁇ (ab10811), ⁇ -histone H3 (ab1791), ⁇ -histone H3K27acetyl (ab4729), and ⁇ -RNA Polymerase II CTD phosphor S2 (ab5095).
  • An sgRNA design tool was used to identify high-scoring candidate sgRNAs to four target regions within and flanking the D4Z4 repeat array ( FIG. 2A ; Table 1). Predicted off-target matches were determined by BLASTing each sequence against the human genomic database (Table 1). High-scoring, non-overlapping candidates with the fewest CpGs and off target matches (four to five sgRNAs for each target region) were cloned individually into BfuAI sites in the pLKO.1-puro U6 sgRNA BfuAI stuffer plasmid and sequence-verified.
  • Myogenic cultures derived from biceps muscle of an FSHD1 patient (17Abic) were used in this example.
  • Patient 17A has two permissive 4qA alleles (-5 repeat units on a contracted 4A161 allele; -26 repeat units on the non-contracted 4A-L161 allele; each 10q allele has ⁇ 37 repeat units).
  • 17Abic myoblasts were grown in Ham's F-10 medium supplemented with 20% FBS (Hyclone), 0.5% chick embryo extract, 1% antibiotics and antimycotics, and 1.2 mmol/l CaCl2.
  • 293T packaging cells were grown in DMEM + 10% FBS + 0.1% penicillin-streptavidin.
  • 293T cells were transfected with lentiviral packaging plasmid (pCMVdR8.91), envelope plasmid (VSV-G), and sgRNA expression plasmid using the TransIT-LT1 transfection reagent (Mirus). Lentiviral supernatants were harvested at 11-hour intervals from 72-108 hours post-transfection. At -70-80% confluency, 17Abic myoblasts were subjected to four serial infections.
  • lentiviral supernatants + 8 ⁇ g/ml polybrene were added to myoblasts and the plates were incubated for 15 minutes at 37 °C, then wrapped well with parafilm before centrifuging for 30 minutes at 1,100 g (32 °C). Following centrifugation, the viral supernatants were replaced with growth medium and cells were allowed to recover for ⁇ 8 hours prior to the next round of infection. Following the last round of infection, cells were switched to differentiation medium (DM) (DMEM/F-12 medium (1:1, Hyclone) plus 2% horse serum (Lonza)) for -40-48 hours prior to harvesting.
  • DM differentiation medium
  • RNAs were extracted using TRIzol (Invitrogen) and purified using the RNeasy Mini kit (Qiagen) after on-column DNase I digestion. Total RNA (2 ⁇ g) was used for cDNA synthesis using Superscript III Reverse Transcriptase (Invitrogen), and 200 ng of cDNA were used for qPCR analysis. Oligonucleotide primer sequences are provided in Table 2.
  • ChIP assays were performed with lentiviral-infected 17Abic differentiated myocytes using the Fast ChIP method. Cells were fixed in 1% formaldehyde in DMEM for 10 minutes and dounced 10x prior to sonication. Cells were sonicated for 12 rounds of 15-second pulses at 65% power output on a Branson Sonifier 450 (VWR Scientific) to shear the DNA to a ladder of -200-800 bp, and efficiency of shearing was verified by agarose gel electrophoresis. Chromatin was immunoprecipitated using 2 ⁇ g of specific antibodies or normal IgG.
  • SYBR green quantitative PCR assays were performed for 40 cycles of: 94 °C for 15 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds. PCR products were analyzed on a 1.5% agarose gel to verify correct size of products and specificity of primer annealing. Oligonucleotide primer sequences are provided in Table 2.
  • Example 2- CRISPR/Cas9 approaches for fascioscapulohumeral muscular dystrophy (FSHD)
  • the CRISPR/dCas9 system was used to test several candidate regions in or flanking the 4q35 D4Z4 / DUX4 locus for the ability to modulate gene expression in the D4Z4 array.
  • Polyadenylated DUX4-fl mRNA levels in FSHD1 myocytes were used as a read-out for gene expression which is specifically derived from the contracted 4q35 D4Z4 array.
  • sgRNAs small guide RNAs
  • dCas9 transcriptional effector platforms are effective in modulating endogenous gene expression levels in mammalian cells.
  • the SunTag system was used, which involves the dual activity of two constructs: (i) dCas9 fused to 10 copies of the GCN4 peptide and (ii) GCN4 antibody fused to the VP64 activator.
  • the dCas9 fused directly to VP64 generally requires multiple, non-overlapping sgRNAs to achieve strong activation of gene expression.
  • the SunTag system allows recruitment of multiple VP64 domains to a single dCas9, resulting in robust gene activation with only a single sgRNA.
  • sgRNAs Single guide RNAs targeting two candidate regions upstream of the D4Z4 repeat ( FIG. 2A ) were produced: the NDE (non-deleted element retained in FSHD patients) sequence and p13-E11, a region distinct in the genome that is used to identify D4Z4 arrays specific to chromosomes 4q35 and 10q26.
  • sgRNAs targeting the promoter, exon 1, and exon 3 of DUX4 were produced.
  • each D4Z4 repeat unit also contains repetitive sequences, and part of the DUX4 exon 1 is duplicated in the NDE, which lies proximal to the array.
  • sgRNA #3-5 targets both the NDE and DUX4 exon 1.
  • sgRNA #6 targets DUX4 intron 2 as well as the DUX4 promoter.
  • sgRNA #6 targets DUX4 intron 2 as well as the DUX4 promoter.
  • sgRNA #6 targets DUX4 intron 2 as well as the DUX4 promoter.
  • four to five sgRNAs were tested for the ability to recruit dCas9-VP64-HA, as assessed by chromatin immunoprecipitation (ChIP) using HA antibodies.
  • ChoIP chromatin immunoprecipitation
  • At least two sgRNAs for each region demonstrated correct targeting of dCas9-VP64-HA (Table 1).
  • VP64 recruitment to the DUX4 promoter or exon 1/NDE yielded robust activation of DUX4-fl in FSHD myocytes ( FIG. 2B , lanes 5-10).
  • sgRNAs #3-5 the transcriptional effector is likely mediating its effects from DUX4 exon 1, and for simplicity, we will refer to these sgRNAs as targeting DUX4 exon 1.
  • FIG. 2B It was observed that when dCas9 lacking a transcriptional effector domain was recruited to DUX4 exon 1, it did not activate DUX4-fl expression ( FIG. 2B , lanes 12-13).
  • dCas9-KRAB Reducing the aberrant expression of DUX4-fl in FSHD by returning the chromatin at the disease locus to a nonpathogenic, repressed state is a viable avenue of therapy. Whether DUX4-fl expression could be reduced in FSHD myocytes using a dCas9-KRAB repressor was examined. When guided by multiple sgRNAs, dCas9-KRAB has proven effective in reducing target gene expression in mammalian cells. Since dCas9-mediated recruitment of VP64 to the DUX4 promoter or exon 1 strongly activated DUX4-fl expression, these regions were selected as candidates for therapeutic targeting with dCas9-KRAB.
  • FSHD myogenic cultures Four serial coinfections of FSHD myogenic cultures were carried out. Cells were infected with various combinations of lentiviral supernatants expressing either dCas9-KRAB or individual sgRNAs targeting the candidate regions. After the final round of infection, the cells were induced to differentiate and harvested ⁇ 40 hours later for analysis of DUX4-fl expression by qRT-PCR.
  • dCas9-KRAB repressor alone had little effect on DUX4-fl levels ( FIG. 3A , lane 2). Consistent with results observed using the SunTag activator system, targeting dCas9-KRAB to either the p13-E11 region or DUX4 exon 3 had no effect on DUX4-fl expression ( FIG. 3A , lanes 3, 8). In contrast to this, targeting dCas9-KRAB to the DUX4 promoter or exon 1 reduced expression of DUX4-fl to -45% of endogenous levels in FSHD myocytes ( FIG. 3A , lanes 4, 6-7).
  • dCas9 effectors often require targeting by multiple, non-overlapping sgRNAs to achieve significant transcriptional modulation, it was observed that in one case, a single sgRNA was effective in reducing DUX4-fl expression ( FIG. 3A , lane 4), and the combination of all six sgRNAs targeting these regions showed no enhanced effect ( FIG. 3A , lane 7).
  • dCas9 can inhibit transcription through steric hindrance of target regions.
  • the effect of a dCas9 variant lacking an effector domain e.g. KRAB was tested.
  • Recruitment of dCas9 alone to any of the target regions did not reduce levels of DUX4-fl ( FIG. 3A , lanes 9-13), demonstrating the importance of the KRAB domain for mediating DUX4-fl repression at the target regions.
  • DUX4-FL expression is restricted to terminally differentiated myocytes.
  • MyHC Myosin heavy chain
  • FRG1 and FRG2 two other FSHD candidate genes that lie proximal to the D4Z4 repeat, were also assayed. Although levels of FRG2 were variable, recruitment of the dCas9 repressor to any of the target regions did not reduce expression of either FRG1 or FRG2 mRNA ( FIG. 3C ).
  • DUX4-FL in FSHD myocytes causes the aberrant upregulation of many downstream targets, including genes expressed in the germline and in early development.
  • TRIM43, ZSCAN4, and MBD3L2 are downstream targets of DUX4-FL that were also found to be upregulated in the myogenic cultures used in this example.
  • expression levels of TRIM43, ZSCAN4, and MBD3L2 were measured by qRT-PCR in the cells.
  • sgRNA #3 expression level of the histone demethylase Jumonji, which contains an off-target match (12-bp seed + PAM) in intron 7, was quantified. It was observed that expression of the untargeted SunTag system alone had a slight repressive effect on levels of Jumonji ( FIG. 5 lane 2). However, targeting the activator with sgRNA #3 did not alter these levels ( FIG. 5 , lane 3). For sgRNA #6, expression levels of the transcription factor KLF14 and the E3 ubiquitin ligase UBR4, which lie 28 and 76 kb downstream of off-target matches (9 bp seed + PAM), were quantified.
  • FSHD myogenic cultures were infected with lentiviral supernatants expressing dCas9-KRAB and sgRNAs targeting the DUX4 promoter or exon 1, and cells were induced to differentiate, then fixed and harvested ⁇ 40 hours later for analysis by ChIP.
  • Recruitment of the dCas9 repressor to the DUX4 promoter resulted in a trend toward increased levels of the KAP1/TRIM28 corepressor, which is recruited by the KRAB domain, as well as HP1 ⁇ and HP1 ⁇ , which are recruited by KAP1 to heterochromatin ( FIGs. 6A-6C , sgRNAs #6-8).
  • FIG. 7 shows data indicating that recruitment of dCas9-KRAB by DUX4 sgRNA represses DUX4-fl expression in vivo.
  • a 30% reduction in DUX4-fl expression level was observed in mice injected with DUX4 sgRNA and dCas9KRAB compared to control mice injected with dCas9KRAB alone.
  • AAV-compatible, catalytically active SpCas9 was recruited to the FSHD locus in primary human myogenic cells by using sgRNA targeting exon 3 of the DUX4 gene.
  • Targeting of the Cas9 nuclease to exon 3 of DUX4 resulted in reduction of DUX4 expression as quantified by relative mRNA expression ( FIG. 8 ).
  • Decreased expression of DUX4 also resulted in reduced relative expression of DUX4 target genes, such as TRIM43 and ZSCAN4, but not the muscle-specific genes MyoG or MyoD ( FIG. 8 ).
  • Table 1 sgRNA sequences targeting the D4Z4/DUX4 region sgRNA (SEQ ID NO:) Target ⁇ 19-nt sequence + PAM ( NGG ) Score ⁇ Enrich ⁇ OT(12) # OT(19) ## 1 p13-E11 TACCACAGACAGCCAACTG GGG 0.61 6.9 18 0 2 p13-E11 TTCACCCAGAACAGTAACT GGG 0.60 1.6 27 0 3 DUX4 E1 CACC CG GGCAAAAGC CG GG AGG 0.61 2.5 8 1 (Y) 4 DUX4 E1 CTGGAAGCACCCCTCAG CG AGG 0.85 2.4 9 3 (14/ 18 /Y) 5 DUX4 E1 CTGGAGGAGCTTTAGGA CG CGG 0.65 2.2 14 6 (14/ 18 /20/22/Y) 6 DUX4 prom CT CG CTCTGGTCTTCTA CG TGG 0.72 2.0 4 0 7 DUX4 prom C CG TC CG

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Dermatology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Claims (10)

  1. In-vitro- oder Ex-vivo-Verfahren zum Hemmen der Expression von DUX4, TRIM43 und ZSCAN4 in einer Zelle, wobei das Verfahren Folgendes umfasst: das Einbringen, in eine Zelle, (i) eines Vektors eines rekombinanten, Adeno-assoziierten Virus (rAAV), der eine für ein katalytisch aktives Cas9-Protein kodierende Nucleinsäure umfasst, und (ii) einer Guide-RNA (gRNA), die spezifisch an eine für eine D4Z4-Makrosatelliten-Wiederholungsregion kodierende Ziel-Nucleinsäuresequenz hybridisiert, in einer Menge, die ausreicht, um die Expression des DUX4-Gens in der Zelle zu hemmen, wobei die gRNA eine Sequenz umfasst, für die eine beliebige der in SEQ ID NO: 9-11 dargelegten Sequenzen kodiert.
  2. Verfahren nach Anspruch 1, wobei die Zelle eine D4Z4-Wiederholungsanordnung mit 11 oder weniger Wiederholungseinheiten umfasst.
  3. Verfahren nach Anspruch 2 oder 3, wobei die Zelle von einem Individuum erhalten wird, das eine fazioskapulohumerale Muskeldystrophie (FSHD) hat oder bei dem ein Risiko dafür besteht.
  4. Verfahren nach einem der Ansprüche 1 bis 3, wobei das katalytisch aktive Cas9-Protein ein Streptococcus-pyogenes-Cas-Protein ist.
  5. Verfahren nach einem der Ansprüche 1 bis 3, wobei das Cas9-Protein die in SEQ ID NO: 42 dargelegte Sequenz umfasst.
  6. Rekombinanter Gen-Editierkomplex zur Verwendung in einem Verfahren zur Behandlung von fazioskapulohumeraler Muskeldystrophie (FSHD) durch Verringern von aberrierender Expression von DUX4, wobei das Verfahren das Verringern der Erkrankungssymptome bei einem bedürftigen Säugetierindividuum umfasst, wobei das Verfahren Folgendes umfasst: das Verabreichen einer therapeutisch wirksamen Menge des rekombinanten Gen-Editierkomplexes an das Individuum, wobei der rekombinante Gen-Editierkomplex (i) einen Vektor eines rekombinanten, Adeno-assoziierten Virus (rAAV), der eine für ein katalytisch aktives Cas9-Protein kodierende Nucleinsäure umfasst, und (ii) eine Guide-RNA (gRNA) umfasst, die spezifisch an eine für eine D4Z4-Makrosatelliten-Wiederholungsregion kodierende Ziel-Nucleinsäuresequenz hybridisiert, in einer Menge, die ausreicht, um die Expression des DUX4-Gens in der Zelle zu hemmen, wobei die gRNA eine Sequenz umfasst, für die eine beliebige der in SEQ ID NO: 9-11 dargelegten Sequenzen kodiert.
  7. Rekombinanter Gen-Editierkomplex zur Verwendung nach Anspruch 6, wobei die Verabreichung durch intramuskuläre Injektion oder intravenöse Injektion erfolgt.
  8. Rekombinanter Gen-Editierkomplex zur Verwendung nach Anspruch 6 oder 7, wobei das Chromosom 4 des Individuums eine D4Z4-Wiederholungsanordnung mit 11 oder weniger Wiederholungseinheiten umfasst.
  9. Rekombinanter Gen-Editierkomplex zur Verwendung nach einem der Ansprüche 6 bis 8, wobei der rekombinante Geneditierkomplex an Muskelzellen des Individuums verabreicht wird, gegebenenfalls an Myoblasten, Myozyten oder enddifferenzierte Muskelzellen des Individuums; oder wobei der rekombinante Geneditierkomplex an eine oder mehrere somatische Zellen des Individuums verabreicht wird, gegebenenfalls an Fibroblasten oder Leukozyten, und wobei die eine oder mehreren somatischen Zellen gegebenenfalls aus dem Individuum isoliert und in eine myogene Linie umprogrammiert sind.
  10. Rekombinanter Gen-Editierkomplex zur Verwendung nach einem der Ansprüche 6 bis 9, wobei das Cas9-Protein die in SEQ ID NO: 42 dargelegte Sequenz umfasst.
EP17853972.2A 2016-09-23 2017-09-22 Stilllegung von dux4 durch rekombinante geneditierungskomplexe Active EP3515506B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662398801P 2016-09-23 2016-09-23
PCT/US2017/052919 WO2018057863A1 (en) 2016-09-23 2017-09-22 Silencing of dux4 by recombinant gene editing complexes

Publications (3)

Publication Number Publication Date
EP3515506A1 EP3515506A1 (de) 2019-07-31
EP3515506A4 EP3515506A4 (de) 2020-04-29
EP3515506B1 true EP3515506B1 (de) 2023-09-13

Family

ID=61691092

Family Applications (1)

Application Number Title Priority Date Filing Date
EP17853972.2A Active EP3515506B1 (de) 2016-09-23 2017-09-22 Stilllegung von dux4 durch rekombinante geneditierungskomplexe

Country Status (4)

Country Link
US (2) US11566237B2 (de)
EP (1) EP3515506B1 (de)
CA (1) CA3074723A1 (de)
WO (1) WO2018057863A1 (de)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11160823B2 (en) * 2016-11-07 2021-11-02 University Of Massachusetts Therapeutic targets for facioscapulohumeral muscular dystrophy
US11674140B2 (en) * 2017-09-07 2023-06-13 The Children's Medical Center Corporation Compositions and methods for treating facioscapulohumeral dystrophy
JP2022516515A (ja) * 2018-12-31 2022-02-28 リサーチ インスティチュート アット ネイションワイド チルドレンズ ホスピタル Ｒｎａ標的化ｃｒｉｓｐｒ－ｃａｓ１３ｂを使用するｄｕｘ４ ｒｎａ発現停止
CN115768487A (zh) * 2020-04-17 2023-03-07 内华达研究与创新公司 用于面肩肱型肌营养不良症的crispr抑制
JP2023539631A (ja) * 2020-08-31 2023-09-15 株式会社モダリス Ｄｕｘ４遺伝子を標的とした顔面肩甲上腕型筋ジストロフィーの治療方法
IL301187A (en) 2020-09-11 2023-05-01 Arrowhead Pharmaceuticals Inc RNAi agents for suppressing DUX4 expression, preparations containing them and uses thereof
CN113337506B (zh) * 2021-06-21 2023-01-03 珠海乐维再生医学科技有限公司 CRISPRi介导的抑制瘢痕形成的外泌体及其制备方法和应用
WO2023018705A1 (en) 2021-08-10 2023-02-16 University Of Massachusetts Compositions and methods for sirna treatment of muscular dystrophy

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012024535A2 (en) 2010-08-18 2012-02-23 Fred Hutchinson Cancer Research Center Methods for determining the presence or risk of developing facioscapulohumeral dystrophy (fshd)
EP3290055A3 (de) * 2011-07-25 2018-05-30 Nationwide Children's Hospital, Inc. Rekombinante virusprodukte und verfahren zur expressionshemmung von dux4
CA2917348A1 (en) * 2013-07-11 2015-01-15 Moderna Therapeutics, Inc. Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use
WO2017180915A2 (en) * 2016-04-13 2017-10-19 Duke University Crispr/cas9-based repressors for silencing gene targets in vivo and methods of use

Also Published As

Publication number Publication date
EP3515506A1 (de) 2019-07-31
CA3074723A1 (en) 2018-03-29
EP3515506A4 (de) 2020-04-29
WO2018057863A1 (en) 2018-03-29
US20230250407A1 (en) 2023-08-10
US11566237B2 (en) 2023-01-31
US20200017842A1 (en) 2020-01-16

Similar Documents

Publication Publication Date Title
US20230250407A1 (en) Silencing of dux4 by recombinant gene editing complexes
US20200392487A1 (en) Excision of retroviral nucleic acid sequences
US10954514B2 (en) Escorted and functionalized guides for CRISPR-Cas systems
CN106061510B (zh) 用于基因组编辑的crispr-cas系统和组合物的递送、用途和治疗应用
JP2020188757A (ja) Ｈｉｖ感染のｒｎａガイド処置のための方法および組成物
CN115175991A (zh) 工程化肌肉靶向组合物
US11963975B2 (en) Therapeutic targets for facioscapulohumeral muscular dystrophy
KR20230053591A (ko) 조작된 근육 표적화 조성물
US20230174958A1 (en) Crispr-inhibition for facioscapulohumeral muscular dystrophy
JP2020505390A (ja) Ｃｒｉｓｐｒ治療薬を送達するためのウイルスベクターとしてのレンチウイルスおよび非組み込みレンチウイルス
US20230383275A1 (en) Sgrna targeting aqp1 rna, and vector and use thereof
US20230119699A1 (en) Diagnostic methods using sirt1 expression
US20240175035A1 (en) Compositions and methods for treating hypercholesterolemia

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20190412

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20200326

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 15/00 20060101ALI20200321BHEP

Ipc: C07H 21/04 20060101ALI20200321BHEP

Ipc: A61K 48/00 20060101AFI20200321BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20210309

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20230403

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230803

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602017074264

Country of ref document: DE

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG9D

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20230913

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20231214

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20231227

Year of fee payment: 7

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20231213

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20231214

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20231227

Year of fee payment: 7

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1610614

Country of ref document: AT

Kind code of ref document: T

Effective date: 20230913

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240113

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240113

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20230913

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20240115

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20231229

Year of fee payment: 7

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230922